This invention relates to methods for the detection of psychoactive compounds in an in vitro neuronal tissue sample, preferably by detecting oscillations of extracellular voltage of before and after the introduction of a candidate sample onto an in vitro neuronal tissue sample and to devices for practicing those processes. Analysis of the extracellular voltage parameters leads to indication of the presence of psychoactive material in the candidate sample and information as to its pharmacological activity and composition. Further, the invention includes a process of initiating and maintaining the presence of repetitive neuronal activity within the in vitro sample. Additionally, this invention includes a method for the stimulation of or initiation of repetitive neuronal activity, e.g., EEG, in an in vitro neuronal tissue sample by introducing a stimulating composition comprising compounds that facilitate or mimic the actions of acetylcholine, serotonin, or catecholamines, such as carbachol.
Rhythmic activity in the hippocampus of small mammals is described as falling into three frequency bands: 4-10 Hz (theta), 10-30 Hz (beta), and above 30 Hz (gamma) (Traub et al., 1998; 1999 for a recent discussion). Theta is by far the best studied of these and is related to, among other things, locomotor activity (Vanderwolf, 1969) and memory encoding (Landfield et al., 1972; Vertes and Kocsis, 1997). In accord with the latter idea is the close relationship between theta and long-term potentiation (Larson and Lynch, 1986; Larson et al., 1986, 1993), a probable substrate of certain forms of memory. The functional correlates of the higher frequency rhythms have recently become the subjects of considerable interest. Gamma activity was first analyzed in the olfactory system (Freeman, 1975 for an early review) with the conclusion that it allows coherence to develop between bulb, piriform cortex, and entorhinal cortex prior to the arrival of an odor (Kay and Freeman, 1998). Activity falling in the gamma range also appears in the visual cortex during cue presentation (Gray and Singer, 1989) where it is proposed to transiently synchronize cells with disparate receptive fields. Synchronization, according to this hypothesis, allows multiple features of a cue to be assembled into a coherent representation (Singer, 1998 for a review). Beta rhythms have not typically been discriminated from the gamma wave in discussions of high frequency hippocampal activity although they have been selectively induced in hippocampal slices (Boddeke et al, 1997). In any event, the growing evidence that high frequency synchronization in essential to coherent operations in the cortical telencephalon has emphasized the importance of identifying the pathways and neurotransmitter systems responsible for the beta and gamma oscillations.
Ascending cholinergic projections promote endogenous oscillations including those in the beta and gamma ranges. Although early work (Stumpf, 1965) found cholinergic blockers or septal lesions to be without obvious effect, subsequent studies showed that fast waves in freely moving rats are enhanced by the cholinesterase inhibitor physostigmine and substantially reduced by antagonists (Leung, 1985). Cholinergic stimulation of hippocampal or entorhinal slices is usually described as inducing seconds-long episodes of theta-like activity (Konopacki et al., 1987; MacVicar and Tse, 1989; Dickson and Alonso, 1997; Williams and Kauer, 1997) but recent experiments show that it can also trigger higher frequency rhythms (20-40 Hz) in cortical and hippocampal slices (Boddeke et al., 1997; Fisahn et al., 1998). Cholinergic. septohippocampal fibers innervate discrete regions of the hippocampal system (Lewis and Shute, 1967; Mosko et al., 1973; Frotscher and Leranth, 1985; Matthews et al., 1987) where they contact subpopulations of interneurons and select dendritic zones of principal cells (Mosko et al., 1973; Lynch et al., 1978; Matthews et al., 1987). Stimulation of muscarinic receptors depolarizes pyramidal cells, depresses release from some interneurons, and increases the excitability of others (Pitler and Alger, 1992; Behrends and Bruggencate, 1993). The combination of in vivo and in vitro results suggests that acetylcholine plays an important role in generating high frequency activity in the cortical telencephalon.
How cholinergically driven high frequency rhythms affect cortical operations depends on whether they are regionally specialized and how they are produced. Carbachol-elicited oscillations are reported to originate in restricted loci in entorhinal cortex (Dickson and Alonso, 1997) and hippocampus (Fisahn et al., 1998) but a more general answer requires systematic mapping over broad expanses of the entorhinohippocampal system. This invention made use of a recently introduced device (Oka et al, 1999) for simultaneously recording from 64 sites to address the origins and regional variations in cholinergically induced high frequency rhythms in the hippocampal cortex.
In addition to the location of the sources, we have found that the oscillations themselves may be studied using various mathematical tools both to identify the presence of psychoactive materials and, in some instances at least, identify and characterize the pharmacological activity or composition of the psychoactive material.
None of the documents discussed above utilize the high frequency oscillations found in the in vitro samples for indication of the presence of, characterization of the pharmacological activity of, or the composition of the psychoactive material.
The inventive methods here include a method for detection of, characterization of the pharmacological activity, and for determining the composition of psychoactive compounds in an in vitro neuronal tissue sample. An allied inventive method is a method for the stimulation of the initiation and stimulation of repetitive neuronal activity variously by the steps of adding a composition that stimulates those oscillations of extracellular voltage, or utilizes a co-implanted bit of neuronal tissue, or stimulates the neuronal sample using an electrical stimulation pattern.
In general, the preferred method for detection of psychoactive compounds in an in vitro neuronal tissue sample includes the steps of inducing and then detecting the presence of oscillations in the tissue sample and providing a baseline value of those oscillations. After that detection, the in vitro sample is brought into contact with a candidate sample of a psychoactive compound or compounds. Coincidentally with (or subsequent to) introduction of the candidate sample, the oscillations, e.g., EEG waves, are then measured. The two sets of oscillation data are then rendered to produce respectively two so-called xe2x80x9ccalculated values.xe2x80x9d Comparing the two calculated values will then allow detection, characterization of the pharmacological activity, and determination of the composition of psychoactive compounds in the sample should one or more be present.
The various oscillations are typically those found in extracellular voltage. For instance, they may be a theta, beta, or gamma EEG waves.
It is desirable to use a multi-electrode dish (xe2x80x9cMEDxe2x80x9d) so that a number of different active or less active sites on the neuronal sample may be simultaneously or sequentially sampled. Use of the MED permits measurement and calculation of spatial relationships; both measured and calculated, amongst the values of the neural oscillations. The multi-electrode nature of the MED also enables the determination and characterization of region-specific effects within the given in vitro neuronal sample.
Appropriate mathematical analysis of the oscillations of extracellular voltage include a Fast Fourier Transform (FFT) of oscillations measured at a single spatial point to enhance differences in amplitude and frequency of the before-and-after single-site measurements.
Similarly, the sequence of oscillations of extra-cellular voltage obtained in an array as a function of time may be subjected to Current Source Density (CSD) analysis to produce and depict current flow patterns within the in vitro neuronal tissue sample.
A further portion of the invention includes the use of chemical or anatomical compositions and electrical stimulation patterns that tend to stimulate or induce repetitive neuronal activity in in vitro neuronal tissue samples. These compositions may be compounds that mimic or facilitate the actions of acetylcholine, serotonin, or catecholamines. They may be co-deposited neuronal tissue. They may be cholinomimetic compounds. A highly desirable chemical compound is carbachol. Electrical stimulations may also be used.
The invention includes various methods of detection, characterization of the pharmacological activity, and determination psychoactive test compound composition. A number of methods for identifying those test compounds are within the methods of the invention.